Receptor-independent role of urokinase-type plasminogen activator in pericellular plasmin and matrix metalloproteinase proteolysis during vascular wound healing in mice

Abstract

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR-/-). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR-/- smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR-/- and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR-/- cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA-mediated plasmin proteolysis to allow cellular migration into a vascular wound.

Figure 1

Schematic representation of the wound healing response to perivascular electric injury and the topographic pattern of neointima formation in u-PAR^+/+ and u-PAR^−/− arteries. The media of an uninjured mouse femoral artery consists of two to three layers of smooth muscle cells. There are no smooth muscle cells in the intima. 2 d after injury, the injured segment in the media is depleted of smooth muscle cells. Within 1 wk after injury, cells begin to repopulate the media and a small neointima is formed at the borders of the injury. The insert shows the presumed migration of smooth muscle cells across the internal elastic lamina, within the media, and alongside the lumen. Within 3–4 wk after injury, the media is repopulated and the neointima has uniformly developed throughout the whole injured region. The vertical lines below each artery denote the equally spaced locations that were used for the topographic analysis in Tables II, III and Fig. 4.

Figure 4

Topographic analysis of the arterial neointima formation in u-PAR^+/+ and u-PAR^−/− arteries within 1 (a) and 3 (b) wk after injury. Within 1 wk after injury, neointima formation initiates from the uninjured borders, whereas within 3 wk after injury, a significant neointima has formed across the entire injured segment in both u-PAR^−/− and u-PAR^+/+ arteries (P = NS versus u-PAR^−/−). The data represent the mean ± SEM of the morphometric measurements in at least seven arteries, determined at similar relative topographic locations throughout the injured segment. The number of the relative locations throughout the injured segment refers to those defined in Fig. 1 beneath each schematically represented artery.

Figure 2

Light microscopic analysis of vascular wound healing and neointima formation after electric injury. All sections were stained with hematoxylin-eosin. (a) uninjured u-PAR^+/+ artery, revealing the presence of smooth muscle cells in the media. (b–d) artery from a u-PAR^+/+ (b) and a u-PAR^−/− (c) mouse (at location 5 as defined in Fig. 1) 3 wk after injury, revealing a similar neointima in u-PAR^−/− as in u-PAR^+/+ arteries. For comparison, an electrically injured artery from a u-PA^−/− mouse (d), revealing impaired wound healing and a much smaller neointima is displayed (reproduced from reference 9). The arrows indicate the internal elastic lamina whereas the arrowheads indicate the external elastic lamina. Bar, 50 μm.

Figure 3

Morphometric analysis in electrically injured arteries, revealing similar cross-sectional neointimal area (a), intima to media ratio (b), percent luminal stenosis (c), and neointimal cell counts (d) in u-PAR^−/− and in u-PAR^+/+ mice after electric injury. The data represent the mean ± SEM of measurements in 8–14 arteries. u-PAR ^+/+, wild-type; u-PAR ^−/−, u-PAR deficient.

Figure 5

Expression of u-PAR. (a–d) In situ hybridization of an uninjured (a and b) or injured (c and d) wild-type artery within 1 wk after injury (at locations 1–3 or 8–10 as defined in Fig. 1) with an antisense (a and c) or sense (b and d) murine u-PAR probe, revealing a low basal level of u-PAR mRNA expression in a quiescent artery and significantly induced u-PAR mRNA levels at the leading front of cellular migration. The arrows indicate the internal elastic lamina; the arrowheads indicate the external elastic lamina. (e–j) Autoradiography (light microscopy: e–h, or electron microscopy: i and j) of cultured u-PAR^+/+ (e, g, and i) and u-PAR^−/− (f, h, and j) smooth muscle cells, incubated with 2 nM murine ^1–48u-PA alone (e, f, i, and j) or together with a 100-fold excess of unlabeled competitor (g and h), revealing significant binding to u-PAR^+/+ but not to u-PAR^−/− smooth muscle cells. Bar, 25 μm.

Figure 6

Ultrastructural localization of u-PA in vivo. Immunogold labeling of u-PA in u-PAR^+/+ (a–c) and u-PAR^−/− (d and e) arteries, revealing u-PA bound to the cell surface (a–c, arrows) on endothelial cells (EC) or smooth muscle cells (SMC), or associated with the extracellular matrix (ECM) (b and c, arrowheads) in u-PAR^+/+ arteries. Note the presence of u-PA gold particles on cell projections (a), and on collagen (CL) fibers in the extracellular matrix (b). In contrast, in u-PAR^−/− arteries, u-PA gold particles were predominantly detected in the pericellular milieu associated with extracellular matrix components (d and e, arrowheads). Note the paucity of u-PA gold particles on the elastin fibers of the internal elastic lamina (IEL) (e). Gold particles labeling u-PA were also found in secretory granules inside u-PAR^−/− cells (d and e, SG).

Figure 7

Immunostaining of MMP-9 and MMP-13. MMP-9 (a and b) and MMP-13 (c and d) immunoreactivity were absent in uninjured wild-type arteries (a and c), but were markedly upregulated throughout the entire vessel wall after injury (b and d). The arrows indicate the internal elastic lamina whereas the arrowheads indicate the external elastic lamina.

Figure 8

Plasmin(ogen) dependent activation of matrix metalloproteinases. (a) gelatin zymography after MMP-9 immunoprecipitation; (b and c) autoradiograph from metabolically labeled macrophage-conditioned medium after MMP-9 (b) or MMP-13 (c) immunoprecipitation. In the absence of plasminogen (−Plg), only the zymogen proMMP-9 (∼92 kD, a and b) and proMMP-13 (∼57 kD, c) are present in the conditioned medium from macrophages of all genotypes. In the presence of plasminogen (+Plg; 10 μg/ml), these proforms are processed to the active MMP-9 (∼82 kD) and MMP-13 (∼45-kD doublet and partially processed forms) by wild-type (WT) and u-PAR–deficient (u-PAR ^−/−) macrophages but not by u-PA– deficient (u-PA ^−/−) macrophages.